We have identified P2X4 receptors (P2X4Rs) as a target for the development of drugs to prevent and/or treat alcohol use disorder (AUD). This hypothesis is derived from compelling systems; genetic, pharmacological and behavioral evidence reporting an inverse relationship between ethanol (EtOH) intake and P2X4R activity. Supporting this hypothesis we found that p2rx4 knock-out mice (P2X4 KO) consumed significantly more EtOH than wildtype (WT) littermate controls. We and others found that ivermectin (IVM), a positive allosteric modula- tor of P2X4Rs, significantly antagonized EtOH inhibition of ATP-gated P2X4Rs and reduced EtOH intake in mice and rats. We have assembled a multidisciplinary team that together will use genetic, molecular, electrophysiological, chemical and behavioral techniques to systematically explore targets in the mesolimbic dopamine (DA) system that will be amenable for drug development. The proposed studies will translate laboratory findings into opportunities to discover and develop novel therapeutics for the prevention and treatment of AUD. Aim 1 studies will test the hypothesis that P2X4Rs within the DA reward system regulate EtOH intake. We will use: in vivo microdialysis coupled with P2X4R modulation (Study 1) and lentiviral- mediated short hairpin RNA (shRNA) knockdown P2X4R expression in the nucleus accumbens (NAc) and/or ventral tegmental area (VTA) (Studies 2 & 3) to gain insights into specific contributions of P2X4Rs in the mesolimbic DA system to EtOH drinking. Male and female P2X4KO and/or WT mice will be tested using two drinking paradigms: (1) 24 hr access, two-bottle choice (free choice) and (2) drinking in the dark (DID; binge EtOH drinking). Aim 2 studies will use a combination of brain slice electrophysiological and knock-out/knock- down technologies to test interrelated hypotheses where we predict that under conditions in which P2X4Rs are reduced, P2X-mediated inhibition of DA neuron firing and EtOH regulation of purinergic inhibition will be reduced or eliminated. This work will provide key information regarding the interaction of P2X4Rs and EtOH, and how it may affect both purinergic and EtOH responses of mesolimbic DA neurons. Aim 3 studies will test the hypothesis that IVM can be used as a platform to identify and develop new compounds that positively modulate P2X4Rs and decrease EtOH-intake. This Aim will be accomplished by designing and synthesizing a series of semisynthetic avermectin and milibemycin analogs followed by their in vitro and in vivo evaluation. Through this iterative process combining synthetic and biological evaluation, we will identify molecules that have maximal anti-alcohol effects while minimizing the IVM-like toxicities (i.e., increased therapeutic index) to yield candidates for further assessment for treating AUD. Taken together, these studies will further our long- term goal of identifying neurochemical mechanisms within key brain reward regions important for regulating EtOH intake. Moreover, this work will set the stage for future translational studies to develop novel pharmacotherapies for AUD.